Vesicle-associated membrane protein 8 (VAMP8), a soluble n-ethylmaleimide-sensitive factor receptor protein, acts as an oncogenic gene in the progression of several malignancies. Nevertheless, the roles and mechanisms of VAMP8 in colorectal cancer (CRC) progression remain unknown. The expression and prognostic significance of VAMP8 in CRC samples were analyzed through bioinformatics analyses. Cell proliferation was detected using CCK-8 and EdU incorporation assays and apoptosis was evaluated via flow cytometry. Western blot analysis was conducted to examine the protein expression. Ferroptosis was evaluated by measurement of iron metabolism, lipid peroxidation, and glutathione (GSH) content. VAMP8 was increased in CRC samples relative to normal samples on the basis of GEPIA and HPA databases. CRC patients with high level of VAMP8 had a worse overall survival. VAMP8 depletion led to a suppression of proliferation and promotion of apoptosis in CRC cells. Additionally, VAMP8 knockdown suppressed beclin1 expression and LC3-II/LC3-I ratio, elevated p62 expression, increased Fe2+, labile iron pool, lipid reactive oxygen species, and malondialdehyde levels, and repressed GSH content and glutathione peroxidase activity. Moreover, VAMP8 knockdown inhibited the activation of janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in CRC cells. Mechanistically, activation of the JAK/STAT3 pathway by JAK1 or JAK2 overexpression attenuated VAMP8 silencing-mediated anti-proliferative, pro-apoptotic, anti-autophagic, and pro-ferroptotic effects on CRC cells. In conclusion, VAMP8 knockdown affects the proliferation, apoptosis, autophagy, and ferroptosis by the JAK/STAT3 pathway in CRC cells.

UNASSIGNED: Vesicle-associated membrane protein 7 (VAMP7) plays oncogenic roles in cancers. However, its clinical significance in breast cancer (BC) tissues remains unknown.
UNASSIGNED: To elucidate the clinical implications of VAMP7, as well as its involvement in the tumor microenvironment and molecular pathways of breast cancer.
UNASSIGNED: BC (n=100) and non-cancerous breast tissues (n= 100) were collected for an immunohistochemical experiment (1:200). The protein expression level of VAMP7 was determined by using a semi-quantitative scoring method. High-throughput RNA-sequencing data of BC tissues were analyzed to confirm the mRNA expression trend of VAMP7. Additionally, the largest BC prognosis cohort data were collected to mine the potential impact VAMP7 has on BC progression. The association between VAMP7 and the microenvironment of BC was evaluated by using a CIBERSORT algorithm. Moreover, we explored the co-expressed molecular mechanisms of VAMP7 in BC by calculating Pearson correlation coefficients and overexpressed genes. Finally, the biological mechanism underlying the relationship between VAMP7 and the key pathways was also explored using gene set enrichment analysis (GSEA). Potential therapeutic strategies were predicted targeting VAMP7.
UNASSIGNED: VAMP7 protein was significantly over-expressed in BC tissue than that in controls (p< 0.001). Compared with 459 normal breast tissues and 113 non-cancerous breast tissues, the expression level of VAMP7 mRNA was significantly increased in 1111 BC tissues. CD4+T cells, macrophages, and naïve B cells had a higher infiltration rate in BC tissues with high VAMP7 expression, while regulatory T cells and CD8+T cells had a lower infiltration rate. Over-expressed VAMP7 was associated with macrophages activation and transition from M1 to M2 polarization. Upregulated VAMP7 could predicted poorer OS, DMFS, PPS, and RFS outcomes. Upregulated VAMP7 co-expressed genes were significantly enriched in the cell cycle checkpoints. GSEA confirmed that over-expressed VAMP7 are markedly associated with functional enrichment in cell cycle related categories, including mitotic spindle, G2M checkpoint, and E2F targets. KU-55933 was predicted as a putative therapeutic drug for BC targeting VAMP7.
UNASSIGNED: VAMP7 was upregulated in BC tissue and correlated with poor prognosis of BC patients. VAMP7 may promote BC progression by targeting the cell cycle pathway.

Autophagy is crucial for degrading and recycling cellular components. Fusion between autophagosomes and lysosomes is pivotal, directing autophagic cargo to degradation. This process is driven by STX17-SNAP29-VAMP8 and STX7-SNAP29-YKT6 in mammalian cells. However, the interaction between STX17 and YKT6 and its significance remain to be revealed. In this study, we challenge the notion that STX17 and YKT6 function independently in autophagosome-lysosome fusion. YKT6, through its SNARE domain, forms a complex with STX17 and SNAP29 on autophagosomes, enhancing autophagy flux. VAMP8 displaces YKT6 from this complex, leading to the formation of the fusogenic complex STX17-SNAP29-VAMP8. We demonstrated that the YKT6-SNAP29-STX17 complex facilitates both lipid and content mixing driven by STX17-SNAP29-VAMP8, suggesting a priming role of YKT6 for efficient membrane fusion. Our results provide a potential regulation mechanism of autophagosome-lysosome fusion, highlighting the importance of YKT6 and its interactions with STX17 and SNAP29 in promoting autophagy flux.

Root growth is sustained by cell division and differentiation of the root apical meristem (RAM), in which brassinosteroid (BR) signaling mediated via the dynamic targeting of BRASSINOSTEROID-INSENSITIVE1 (BRI1) plays complex roles. BRI1 is constitutively secreted to the plasma membrane (PM), internalized, and recycled or delivered into vacuoles, whose PM abundance is critical for BR signaling. Vesicle-target membrane fusion is regulated by heterotetrameric SNARE complexes. SNARE proteins have been implicated in BRI1 targeting, but how SNAREs affect RAM development is unclear. We report that Arabidopsis (Arabidopsis thaliana) YKT61, an atypical R-SNARE protein, is critical for BR-controlled RAM development through the dynamic targeting of BRI1. Functional loss of YKT61 is lethal for both male and female gametophytes. By using weak mutant alleles of YKT61, ykt61-partially complemented (ykt61-pc), we show that YKT61 knockdown results in a reduction of RAM length due to reduced cell division, similar to that in bri1-116. YKT61 physically interacts with BRI1 and is critical for the dynamic recycling of BRI1 to the PM. We further determine that YKT61 is critical for the dynamic biogenesis of vacuoles, for the maintenance of Golgi morphology, and for endocytosis, which may have a broad effect on development. Endomembrane compartments connected via vesicular machinery, such as SNAREs, influence nuclear-controlled cellular activities such as division and differentiation by affecting the dynamic targeting of membrane proteins, supporting a retro-signaling pathway from the endomembrane system to the nucleus.

Chemotherapeutic resistance is one of the most common reasons for poor prognosis of patients with nasopharyngeal carcinoma (NPC). We found that CENPN can promote the growth, proliferation and apoptosis resistance of NPC cells, but its relationship with chemotherapeutic resistance in NPC is unclear. Here we verified that the CENPN expression level in NPC patients was positively correlated with the degree of paclitaxel (PTX) resistance and a poor prognosis through analysis of clinical cases. VAMP8 expression was significantly increased after knockdown of CENPN by transcriptome sequencing. We found in cell experiments that CENPN inhibited macroautophagy/autophagy and VAMP8 expression and significantly increased PTX resistance. Overexpression of CENPN reduced the inhibitory effects of PTX on survival, cell proliferation, cell cycle progression and apoptosis resistance in NPC cells by inhibiting autophagy. In turn, knockdown of CENPN can affect the phenotype of NPC cells by increasing autophagy to achieve PTX sensitization. Sequential knockdown of CENPN and VAMP8 reversed the PTX-sensitizing effect of CENPN knockdown alone. Experiments in nude mice confirmed that knockdown of CENPN can increase VAMP8 expression, enhance autophagy and increase the sensitivity of NPC cells to PTX. Mechanistic studies showed that CENPN inhibited the translocation of p-CREB into the nucleus of NPC cells, resulting in the decreased binding of p-CREB to the VAMP8 promoter, thereby inhibiting the transcription of VAMP8. These results demonstrate that CENPN may be a marker for predicting chemotherapeutic efficacy and a potential target for inducing chemosensitization to agents such as PTX.Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; CENPN: centromere protein N; CQ: chloroquine; CREB: cAMP responsive element binding protein; ChIP: chromatin immunoprecipitation assay; IC50: half-maximal inhibitory concentration; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NPC: nasopharyngeal carcinoma; NPG: nasopharyngitis; oeCENPN: overexpressed CENPN; PTX: paclitaxel; RAPA: rapamycin; RNA-seq: transcriptome sequencing; shCENPN: small hairpin RNA expression vector targeting the human CENPN gene; shCENPN-shVAMP8: sequential knockdown targeting the human CENPN gene and VAMP8 gene; shVAMP8: small hairpin RNA expression vector targeting the human VAMP8 gene; TEM: transmission electron microscopy; TIR: tumor inhibitory rate; VAMP8: vesicle associated membrane protein 8.

Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable RT‒qPCR gene expression analysis results. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on their expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder, and RefFinder under different conditions and developmental stages. The results revealed that MSF1 domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA-binding protein 1 (TBP1), and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study on the identification of reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.

Osteosarcoma is a highly metastatic malignant bone tumor, necessitating the development of new treatments to target its metastasis. Recent studies have revealed the significance of VAMP8 in regulating various signaling pathways in various types of cancer. However, the specific functional role of VAMP8 in osteosarcoma progression remains unclear. In this study, we observed a significant downregulation of VAMP8 in osteosarcoma cells and tissues. Low levels of VAMP8 in osteosarcoma tissues were associated with patients\' poor prognosis. VAMP8 inhibited the migration and invasion capability of osteosarcoma cells. Mechanically, we identified DDX5 as a novel interacting partner of VAMP8, and the conjunction of VAMP8 and DDX5 promoted the degradation of DDX5 via the ubiquitin-proteasome system. Moreover, reduced levels of DDX5 led to the downregulation of β-catenin, thereby suppressing the epithelial-mesenchymal transition (EMT). Additionally, VAMP8 promoted autophagy flux, which may contribute to the suppression of osteosarcoma metastasis. In conclusion, our study anticipated that VAMP8 inhibits osteosarcoma metastasis by promoting the proteasomal degradation of DDX5, consequently inhibiting WNT/β-catenin signaling and EMT. Dysregulation of autophagy by VAMP8 is also implicated as a potential mechanism. These findings provide new insights into the biological nature driving osteosarcoma metastasis and highlight the modulation of VAMP8 as a potential therapeutic strategy for targeting osteosarcoma metastasis.

Ykt6 is one of the most conserved SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) proteins involved in multiple intracellular membrane trafficking processes. The membrane-anchoring function of Ykt6 has been elucidated to result from its conformational transition from a closed state to an open state. Two ways of regulating the conformational transition were proposed: the C-terminal lipidation and the phosphorylation at the SNARE core. Despite many aspects of common properties, Ykt6 displays differential cellular localizations and functional behaviors in different species, such as yeast, mammals, and worms. The structure-function relationship underlying these differences remains elusive. Here, we combined biochemical characterization, single-molecule FRET measurement, and molecular dynamics simulation to compare the conformational dynamics of yeast and rat Ykt6. Compared to rat Ykt6 (rYkt6), yeast Ykt6 (yYkt6) has more open conformations and could not bind dodecylphosphocholine that inhibits rYkt6 in the closed state. A point mutation T46L/Q57A was shown to be able to convert yYkt6 to a more closed and dodecylphosphocholine-bound state, where Leu46 contributes key hydrophobic interactions for the closed state. We also demonstrated that the phospho-mutation S174D could shift the conformation of rYkt6 to a more open state, but the corresponding mutation S176D in yYkt6 leads to a slightly more closed conformation. These observations shed light on the regulatory mechanism underlying the variations of Ykt6 functions across species.

Fungi are an important group of microorganisms that play crucial roles in a variety of ecological and biotechnological processes. Fungi depend on intracellular protein trafficking, which involves moving proteins from their site of synthesis to the final destination within or outside the cell. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are vital components of vesicle trafficking and membrane fusion, ultimately leading to the release of cargos to the target destination. The v-SNARE (vesicle-associated SNARE) Snc1 is responsible for anterograde and retrograde vesicle trafficking between the plasma membrane (PM) and Golgi. It allows for the fusion of exocytic vesicles to the PM and the subsequent recycling of Golgi-localized proteins back to the Golgi via three distinct and parallel recycling pathways. This recycling process requires several components, including a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), a retromer submit, and the COPI coat complex. Snc1 interacts with exocytic SNAREs (Sso1/2, Sec9) and the exocytic complex to complete the process of exocytosis. It also interacts with endocytic SNAREs (Tlg1 and Tlg2) during endocytic trafficking. Snc1 has been extensively investigated in fungi and has been found to play crucial roles in various aspects of intracellular protein trafficking. When Snc1 is overexpressed alone or in combination with some key secretory components, it results in enhanced protein production. This article will cover the role of Snc1 in the anterograde and retrograde trafficking of fungi and its interactions with other proteins for efficient cellular transportation.

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.